Whole-cell voltage-clamp and single channel recording techniques are used to study drug interactions with voltage-activated and ligand-activated ion channels in cultured neurons and in heterologous cells transfected with cloned ion channel subunit genes. The aim of this work is to explore new strategies for the rational development of anti-epileptic drugs based upon their interaction with neuronal ion channel systems. During the present reporting period, an analysis was carried out of NMDA receptor channel block by the enantiomers of BIII 277 CL (2R-[2alpha3 (R*),6alpha]-3-(2- methoxypropyl)-6, 11, 11 -trimethyl-2,6-methano- 1,2,3,4,5,6-hexahydro-3- benzazocin-9-ol hydrochloride), a benzomorphan with anticonvulsant and neuroprotective properties that exhibits significantly lower toxicity than other channel blocking NMDA receptor antagonists. A novel model was developed to explain the interaction of this drug with NMDA receptors. In addition, studies were conducted examining the subunit specificity of ADCI for block of NMDA receptors (see report for Project Number Z01 NS 02772-10 ERB). BIII 277 CL and BIII 281 CL produced a slow use-dependent block of whole- cell NMDA receptor currents in cultured hippocampal neurons. Once block was established, recovery occurred slowly. The steady-state IC 50 values derived from logistic fits to concentration-block isotherms obtained at approximately 60 mV were 5.3 and 64nM, respectively. The benzomorphans had no effect on currents evoked by AMPA and GABA; but minimally inhibited kainate-evoked currents at high concentrations. BIII 277 CL and BIII 281 CL failed to bind and block closed NMDA receptor channels, and the block was occluded by magnesium, consistent with an open channel blocking mechanism. Steady-state block was diminished by depolarization; analysis of the voltage-dependence of block indicated that BIII 281 CL binds within the channel at an electrical depth of 0.46. Recordings of single NMDA receptor channels in outside-out membrane patches confirmed the slow, persistent blocking action obtained in whole cell recordings. In addition, at high concentrations, flickering of the unitary currents was observed consistent with a low affinity channel blocking action. Taking the present data together with previously obtained structure-activity information for N-substituted benzomorphans, a three-mode blocking model was developed in which there are three interaction sites for binding of the high affinity ligand BIII 277 CL [possibly, (i) protonated nitrogen, (ii) methoxypropyl side chain and (iii) tetralin ring]. In this model, the drug can bind in one of three modes by docking at one, two or all three interaction points, but cannot transition between modes. The model further proposes that the lower affinity enantiomer BIII 281 CL binds in modes with one and two but not all three interaction points docked. It was concluded that BIII 277 CL and BIII 281 CL are potent and selective, use-dependent (uncompetitive) channel blocking NMDA receptor antagonists. The substantially higher affinity that BIII 277 CL exhibits for the NMDA receptor in comparison with its enantiomer and other benzomorphans appears to be due to stabilization of binding at three sites within the channel. BIII 281 CL serves as a prototype upon which to base the development of low toxicity channel blocking NMDA receptor antagonists for the treatment of epilepsy and other neurological conditions associated with excessive activation of NMDA receptors, including brain ischemia and some forms of neurodegeneration.